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REVIEW published: 31 May 2016 doi: 10.3389/fmicb.2016.00744

The Ecology of Acidobacteria: Moving beyond Genes and Genomes

Anna M. Kielak1†, Cristine C. Barreto2†, George A. Kowalchuk3, Johannes A. van Veen1 and Eiko E. Kuramae1*

1 Department of Microbial Ecology, The Netherlands Institute of Ecology – Koninklijke Nederlandse Akademie van Wetenschappen, Wageningen, Netherlands, 2 Graduate Program in Genomic Sciences and Biotechnology, Universidade Católica de Brasília, Brasília, Brazil, 3 Ecology and Biodiversity Group, University of Utrecht, Utrecht, Netherlands

The phylum Acidobacteria is one of the most widespread and abundant on the planet, yet remarkably our knowledge of the role of these diverse organisms in the functioning of terrestrial ecosystems remains surprisingly rudimentary. This blatant knowledge gap stems to a large degree from the difficulties associated with the cultivation of these bacteria by classical means. Given the phylogenetic breadth of the Acidobacteria, which is similar to the metabolically diverse Proteobacteria, it is clear that detailed and functional descriptions of acidobacterial assemblages are necessary. Fortunately, recent advances are providing a glimpse into the ecology of members of the phylum Acidobacteria. These include novel cultivation and enrichment strategies, genomic

Edited by: characterization and analyses of metagenomic DNA from environmental samples. Here, Rich Boden, we couple the data from these complementary approaches for a better understanding University of Plymouth, UK of their role in the environment, thereby providing some initial insights into the ecology of Reviewed by: Stephanie A. Eichorst, this important phylum. All cultured acidobacterial type species are heterotrophic, and University of Vienna, Austria members of subdivisions 1, 3, and 4 appear to be more versatile in carbohydrate Hinsby Cadillo-Quiroz, utilization. Genomic and metagenomic data predict a number of ecologically relevant Arizona State University, USA capabilities for some acidobacteria, including the ability to: use of nitrite as N source, *Correspondence: Eiko E. Kuramae respond to soil macro-, micro nutrients and soil acidity, express multiple active [email protected] transporters, degrade gellan gum and produce exopolysaccharide (EPS). Although †These authors have contributed these predicted properties allude to a competitive life style in soil, only very few of these equally to this work. prediction shave been confirmed via physiological studies. The increased availability of

Specialty section: genomic and physiological information, coupled to distribution data in field surveys and This article was submitted to experiments, should direct future progress in unraveling the ecology of this important Terrestrial Microbiology, a section of the journal but still enigmatic phylum. Frontiers in Microbiology Keywords: Acidobacteria, carbohydrate metabolism, transporters, nitrogen metabolism, EPS, soil factors Received: 08 October 2015 Accepted: 03 May 2016 Published: 31 May 2016 INTRODUCTION Citation: Kielak AM, Barreto CC, Although the Acidobacteria were only recognized as a phylum relatively recently, their abundance Kowalchuk GA, van Veen JA across a range of ecosystems, especially soils, has demanded research into their ecology. 16S rRNA and Kuramae EE (2016) The Ecology gene-based approaches as well as environmental shotgun metagenomic analyses have revealed of Acidobacteria: Moving beyond Genes and Genomes. that the Acidobacteria represent a highly diverse phylum resident to a wide range of habitats Front. Microbiol. 7:744. around the globe (Chow et al., 2002; Kuske et al., 2002; Gremion et al., 2003; Quaiser et al., 2003; doi: 10.3389/fmicb.2016.00744 Fierer et al., 2005; Stafford et al., 2005; Janssen, 2006; Sanguin et al., 2006; de Carcer et al., 2007;

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Kim et al., 2007; Singh et al., 2007; DeAngelis et al., 2009; Jesus Acidobacteria phylum. Based on phylogenetic analysis of 16S et al., 2009; Kielak et al., 2009; Navarrete et al., 2010, 2013b; Zhang rRNA gene sequences, the Acidobacteria phylum raised from et al., 2014). However, despite their high abundance and diversity, the originally described four to six subdivisions (Kuske et al., we still have relatively little information regarding the actual 1997; Ludwig et al., 1997; Barns et al., 1999, 2007) to over eight activities and ecology of members of this phylum, a shortcoming subdivisions in 1998 (Hugenholtz et al., 1998) and in 2005 that can be attributed to a large extent to the difficulties in this number increased to 11 (Zimmermann et al., 2005) deeply cultivating the majority of acidobacteria and their poor coverage branching and strongly supported subdivisions. Currently there in bacterial culture collections (Bryant et al., 2007; Lee et al., 2008; are 26 accepted subdivisions (Barns et al., 2007) in the Ribosomal da Rocha et al., 2009; Eichorst et al., 2011; Navarrete et al., 2013b). Database Project. The first recognized strain and species of the However, environmental surveys have provided insight into some phylum Acidobacteria was Acidobacterium capsulatum obtained the environmental factors that may drive acidobacteria dynamics, from an acid mine drainage in Japan (Kishimoto and Tano, such as pH and nutrients (Fierer et al., 2007; Jones et al., 2009; 1987; Kishimoto et al., 1991; Abed et al., 2002). Although the Lauber et al., 2009; Navarrete et al., 2013b). second isolate belonging to this phylum was Holophaga foetida In 2009, the first sequenced genomes of acidobacteria strains first described in 1994, it was not initially recognized as related became available, providing preliminary genetic insights into to Acidobacteria capsulatum. Instead, it was thought to belong the potential physiology and environment functions of several to the phylum Proteobacteria (Liesack et al., 1994). A few years members of this phylum (Ward et al., 2009). In these first later, a closely related bacterium named Geothrix fermentans genomic studies, five aspects of physiological received particular was isolated (Coates et al., 1999) and subsequently another attention: (i) carbon usage, (ii) nitrogen assimilation, (iii) closely related bacterium Acanthopleuribacter pedis, the first metabolism of iron, (iv) antimicrobials, and (v) abundance acidobacteria obtained from a marine sample, was described of transporters. Besides genome sequencing of cultivated (Fukunaga et al., 2008). Since these isolates were very distantly isolates, addition information regarding genomic properties of related to A. capsulatum, it was proposed that it should be acidobacteria has been derived from metagenomics studies (Liles included in a new class named Holophagae. Acidobacteriia et al., 2003; Quaiser et al., 2003, 2008; Riaz et al., 2008; Jones et al., and Holophagae are the only classes currently included in the 2009; Kielak et al., 2010; Parsley et al., 2011; Faoro et al., 2012; most recent edition of the Bergey’s Manual (Thrash and Coates, Navarrete et al., 2013b; Mendes et al., 2014). 2014). In this review, we couple the complementary data coming Currently Acidobacteria phylum has 26 subdivisions based from physiological, genomic and metagenomics studies to seek on the extremely broad diversity of acidobacterial populations a better understanding of the role of Acidobacteria in the found in uranium-contaminated soils (Barns et al., 2007). Newly environment, thereby providing some initial insights into the characterized acidobacteria from subdivision 1 may challenge ecology of this important phylum. We aim to not only give a more this taxonomy in the near future, since of 16S rRNA gene analysis complete picture of the current knowledge of Acidobacteria, but has consistently shown that the genera Acidobacteria,‘Acidipila’ also seek to provide a solid base for future experiments geared (Okamura et al., 2011), Telmatobacter (Pankratov et al., 2012), toward gaining a better understanding of the ecological roles and Acidicapsa (Kulichevskaya et al., 2012) form a group that is played by members of this phylum. distinct from the genera Granulicella, Terriglobus, Bryocella, and Edaphobacter (Eichorst et al., 2007; Koch et al., 2008; Pankratov and Dedysh, 2010; Männistö et al., 2011, 2012; Rawat et al., HISTORY AND GENERAL INFORMATION 2012a,b; Baik et al., 2013; Whang et al., 2014). ON THE PHYLUM Acidobacteria The vast majority of isolates cultivated to date are affiliated with acidobacteria subdivision 1 (Class Acidobacteriia). They are The introduction of molecular biological strategies into microbial all heterotrophic, most species are aerobic or microaerophilic ecology over the past decades has yielded a new perspective on and some species (Telmatobacter bradus, Acidobacterium the breadth and vastness of microbial diversity. The phylum capsulatum) are facultative anaerobic bacteria (Pankratov of the Acidobacteria is one of the bacterial lineages that has et al., 2012). Members of subdivisions 3, 4, 8 (currently profited most from the cultivation-independent interrogation Class Holophagae), 10, and 23 are heterotrophic as well. of environmental samples. Indeed, in the past two decades, Thermotomaculum (subdivision 10) and Thermoanaerobaculum this phylum has grown from being virtually unknown to being (subdivision 23) are thermophilic anaerobic bacteria (Izumi et al., recognized as one of the most abundant and diverse on Earth. 2012; Losey et al., 2013). Chloracidobacterium thermophilum This phylum is particularly abundant in soil habitats that can is photoheterotrophic (Bryant et al., 2007; Tank and Bryant, represent up to 52% from the total bacterial community (Dunbar 2015) and Pyrinomonas methylaliphatogenes can consume H2 et al., 2002; Sait et al., 2002) and averaging approximately 20% (Greening et al., 2015), both from subdivision 4. Subdivision of the microbial community across diverse soil environments 8 contains one aerobic (Acanthopleuribacter) and two strictly (Janssen, 2006). anaerobic isolates (Holophaga and Geothrix). There are reports Although 16S rRNA gene sequences related to the of acidobacteria isolates belonging to subdivisions 2 and 6, Acidobacteria were obtained as early as 1993 (Stackebrandt but they still do not have valid taxonomic names (Sait et al., et al., 1993) it was only in 1997 that they were associated 2002; George et al., 2011; Parsley et al., 2011). Subdivisions 1 with sequences belonging to cultured members of the current and 3 of the phylum Acidobacteria together with thermophilic

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Thermoanaerobacter species are capable of biosynthesizing total acidophilic strains belonging to subdivision 1 (Sait et al., 2006). fatty acids lipid (Damste et al., 2011). Currently, there a total of This combination of strategies seems to enrich not only for 40 species belonging to 22 genera: eleven genera of subdivision 1, Acidobacteria but for many other groups of slow-growing two of subdivision 3; four of subdivision 4, three of subdivision bacteria. The association of a molecular technique such as 8, one of subdivision 10, and one of subdivision 23 (Figure 1). the high-throughput plate-wash PCR (Stevenson et al., 2004) In addition, there are the genome sequences of Koribacter and or colony-PCR (de Castro et al., 2013), both using phylum- Solibacter, but there is little information on their physiology. specific 16S rRNA gene primers has improved the screening and identification of colonies belonging to the Acidobacteria subgroup 1. Once Acidobacteria isolation under low nutrient THE IMPACT OF NEW ISOLATION conditions is achieved, strains can often be transferred to richer METHODS media (e.g., TSA and R2A) for more convenient propagation (de Castro et al., 2013). Despite the importance of these recent Changes in the traditional methods for culturing bacteria from advances in cultivation methods, further improvements are soils have significantly improved the isolation of Acidobacteria clearly needed since only eight of a total of 26 subdivisions are strains in recent years. These new strategies involve the use known to have representatives in culture. A large number of of relatively low concentration of nutrients, non-traditional Acidobacteria isolates have been recovered from the Australian sources of carbon or complex polysaccharides (Janssen et al., soil Ellin bank (Janssen et al., 2002; Sait et al., 2002; Davis et al., 2002; Sait et al., 2002; Pankratov et al., 2008; Eichorst et al., 2005). These studies were important during the development 2011), longer periods of incubation, the use of gellan gum as of new strategies for culturing soil bacteria, and two of solidifying agent, (Janssen et al., 2002; Sait et al., 2002), non- these isolates, ‘Koribacter versatilis’ Ellin345 (CP000360) and standard CO2 atmospheric conditions of incubation, addition of ‘Solibacter usitatus’ Ellin6076 (CP000473), were used in the quorum-signaling molecules, catalase or cations (Stevenson et al., first Acidobacteria genome investigations (Ward et al., 2009). 2004; George et al., 2011; Navarrete et al., 2013a), amendments However, many of these bacteria have not yet been fully of inhibitors for undesired organisms, and amendment of characterized and still do not possess valid taxonomical names. environmental extracts in growth media (Foesel et al., 2013). Further, micro-cultivation strategies combined with single-cell It is suggested that raising the CO2 concentration may not sequencing should provide access to new acidobacterial genomes, only better mimic the CO2 concentrations typically found in and in turn this genomic information may help to inform soils, but may also decrease medium pH, thereby benefiting future isolation efforts as cultivation is still required for most certain members of the acidobacteria, especially moderate physiological characterizations.

FIGURE 1 | Dendrogram showing the general characteristics of Acidobacteria type species, only. The dendrogram was obtained using nearly-full 16S rRNA gene sequences obtained and aligned using the Ribosomal Database Project. Dendrogram were obtained with MEGA7 (Tamura et al., 2011) using the Neighbor-joining method (Saitou and Nei, 1987); bootstrapping values were based on 1000 repetitions and are shown next to the branches (Felsenstein, 1985). There were a total of 1343 positions in the final dataset. 1. Acidobacterium capsulatum was originally described as an aerobic bacteria, but later it was demonstrated a weak anaerobic growth by fermentation (Pankratov et al., 2012). 2. H. foetida is homoacetogen and G. fermentans is fermentative. ND, not determined. Quote marks (‘) indicate that this species is deposited in a culture collection, but its name does not retain its validity and standing in nomenclature.

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THE IMPACT OF ACIDOBACTERIA able to use chitin as a carbon source (Foesel et al., 2013; Huber GENOMES AND LINKS TO et al., 2014)(Figure 2). Most of the Acidobacteria subdivisions 1, 3, or 4 examined to date is unable to use carboxymethyl PHYSIOLOGICAL STUDIES cellulose, but there is evidence that Aridibacter kavangonensis (subdivision 4) is able to utilize micro-crystalline cellulose The first comparative genome analysis between of A. capsulatum (Huber et al., 2014). Although it is still premature to draw and two bacteria originated from the Ellin collection, ‘S. usitatus’ general conclusions related to the degradation of these abundant Ellin6076 (subdivision 1), and ‘K. versatilis’ Ellin345 (subdivision polysaccharides by Acidobacteria in nature, xylan degradation 3) provided numerous insights into the physiology of members has been broadly demonstrated, which may play a role in plant of the Acidobacteria (Ward et al., 2009; Challacombe et al., cell wall degradation (Pankratov et al., 2008; Eichorst et al., 2011). 2011). Since then, the number of acidobacterial genomes being The discrepancies between genome predictions and observed sequenced remains rather limited. Currently, there are 10 activities may stem from our ability to provide cultivation published genomes of Acidobacteria available: five subdivision conditions that lead to the expression of the target activities. 1 (Ward et al., 2009; Rawat et al., 2012a,b, 2013, 2014), one Alternatively, current automatic genome annotation pipelines subdivision 3 (Ward et al., 2009), two subdivision 4 (Costas et al., may not successfully differentiate genes involved for instance 2012; Lee et al., 2015), and one subdivision 8 – class Holophagae in chitin and cellulose degradation from genes involved (Anderson et al., 2012); 1 subdivision 23 (Stamps et al., 2014). in the degradation of other glycosyl hydrolases, such as Below, we summarize some of the major findings revealed via xylan. Systematic studies on the degradation of cellulose by the currently available acidobacterial genome sequences linked to Acidobacteria grown on different culture conditions may help to physiological studies. test the hypothesis of gene regulation by sugars present in the media, for example. On the other hand, it has been reported that CARBOHYDRATE METABOLISM in bacteria many genes involved in cellulose degradation may be involved in the infection of plant cells or in the synthesis of Among all the physiological aspects revealed by genomics, bacterial cellulose (Koeck et al., 2014). carbohydrate metabolism has been studied most widely, which Enzymatic activities observed in Acidobacteria have usually is not surprising considering that carbon usage is one of the been detected using commercial kits with chromogenic physiological requirements for the description of new species substrates. Members of subdivision 1 possess a broader range in taxonomic studies. At least one species of each of the eight of enzymes related to sugar usage than those from other recognized genera of subdivision 1 is able to use D-glucose, D- subdivisions (Figure 3; Supplementary Table S1). Galactosidases xylose, and lactose as carbon sources (Figure 2). The ability to are enzymes involved in the hydrolysis of galactose-containing use glucose and xylose makes sense given the fact that cellulose sugars, while beta galactosidades are involved in the degradation or xylan are often the major carbon sources in the culture media of lactose. Since all genera of Acidobacteria subdivision 1 are most typically used for the isolation of Acidobacteria. In addition, able to use lactose, it is not surprising to find this enzyme these bacteria were able to use most of the tested oligosaccharides, included in their enzymatic profile. Glucosidases are involved although maltose and cellobiose were not able to support growth in the degradation of polysaccharides, especially cellulose of Edaphobacter species. Interestingly, the majority of subdivision and starch. Although starch is used by most Acidobacteria 1 species were unable to use fucose or sorbose, carbohydrates that (Figure 2), cellulose degradation has not yet been unequivocally are only minor components of plant cell walls and rather scarce demonstrated for most Acidobacteria, as explained above. in soil (Li et al., 2013). Interestingly, β-glucosidase has successfully been purified and Although several acidobacterial genomes have been shown characterized from A. capsulatum (Abed et al., 2002). This to contain genes encoding for the degradation of different enzyme was induced by the presence of cellobiose in the medium, polysaccharides (Figure 2), experimental data on the use of and it was active on p-nitrophenyl-p-D-glucopyranoside (100%), polysaccharides generally do not support genomic predictions. cellobiose (39%), and β-gentiobiose (33%). In a subsequent study, At least 50% of the genera have members able to use starch, the gene xynA for a endo-β-1,4-xylanase from A. capsulatum lamminarin, and xylan. In contrast, chitin usage has not yet (Koch et al., 2008) was cloned and expressed in Escherichia coli been demonstrated for any member of Acidobacteria subdivision (Dedysh et al., 2012). This enzyme was demonstrated to be active 1. Similarly, cellulose was another substrate predicted to be on xylan and cellobiose (100% of relative activity), but has low degraded by Acidobacteria genome annotation. However, only activity on CM-cellulose (5.4%) and no activity on filter paper of Telmatobacter bradus (subdivision 1) has been demonstrated Avicel. to be able to use crystalline cellulose (Pankratov et al., 2012) Janssen et al.(2002) were the pioneers to use gellan gum and Edaphobacter cerasi (Yamada et al., 2014) is able to as solidifying agent in culture medium used for Acidobacteria grow on CM-cellulose. Terracidiphilus gabretensis produces isolation. In contrast to agar, which is obtained from seaweed, extracellular enzymes implicated in the degradation of plant- gellan gum is a substrate that is produced (and degraded) by soil derived biopolymers what was confirmed by genome analysis bacteria. At least two species of Acidobacteria have demonstrated by the presence of enzymatic machinery required for organic ability to use gellan gum: Telmatobacter bradus and Bryocella matter decomposition (Garcia-Fraile et al., 2016). In contrast to elongata. One known pathway for the degradation of gellan- Acidobacteria from subdivision 1, members of subdivision 4 are gum involves the action of gellan lyases, α-L-rhamnosidases,

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FIGURE 2 | Usage of carbon sources by Acidobacteria in culture-based experiments with type strain species. A positive score was recorded if at least one species within a genus is able to use a respective sugar. (A) Acidobacteria subdivision 1. (B) Acidobacteria subdivisions 3 and 4. Usage of carbon source obtained from original references that described each of the type species, in order of publication: Kishimoto et al., 1991; Liesack et al., 1994; Coates et al., 1999; Eichorst et al., 2007; Fukunaga et al., 2008; Koch et al., 2008; Kulichevskaya et al., 2010, 2012, 2014; Pankratov and Dedysh, 2010; Männistö et al., 2011, 2012; Okamura et al., 2011; Dedysh et al., 2012; Izumi et al., 2012; Pankratov et al., 2012; Baik et al., 2013; Foesel et al., 2013, 2016; Losey et al., 2013; Crowe et al., 2014; Huber et al., 2014; Whang et al., 2014; Yamada et al., 2014; Pascual et al., 2015; Tank and Bryant, 2015; Garcia-Fraile et al., 2016; Jiang et al., 2016; Llado et al., 2016).

unsaturated glucuronyl hydrolases, and β-D-glucosidases (Baik genomes (Ward et al., 2009). Nitrate reduction has been et al., 2013). Members of Acidobacteria subdivision 1 were investigated in almost all members of subdivision 1, with reported to be able to use the monosaccharides rhamnose and the exception of Acidobacterium and Acidicapsa. Among all glucose. Also, at least one β-D-glucosidase was characterized Granulicella species, G. mallensis was reported to perform nitrate from A. capsulatum, which may be involved in gellan gum reduction (Männistö et al., 2012). A. rosea and B. elongata are degradation. However, the in silico comparison of 10 available also able to reduce nitrate to nitrite. Among other subdivisions, genomes, offered no evidence of gellan lyase (EC 4.2.2.25) only Geothrix fermentans subdivision 8 was shown to be able genes for gellan lyase usage. Moreover, Naumoff and Dedysh to reduce nitrate. This organism is an iron reducer that can (2012) reported that the presence of α-rhamnosidases is not use nitrate as an alternative electron acceptor. All of these a phylogenetically determined trait, but that this function was Acidobacteria were able to use yeast extract that, in addition to obtained by lateral gene transfer from Bacteroidetes and in the ammonium, may be a preferred nitrogen source (Coates et al., case of Holophaga foetida from a fungus. The investigation of the 1999). The presence of the nirA gene, which encodes nitrate enzymatic pathway for gellan gum degradation may merit further reductase, also appears to be limited to subdivision 1, suggesting investigation, since this is a bacterial polysaccharide. Therefore, that members of this subdivision may reduce nitrate to nitrite by in addition to the possible metabolism of polysaccharides derived the assimilatory pathway, which is further reduced to ammonia from plants, the usage of gellan gum suggests an interaction with and assimilated into glutamate. Nevertheless, the direct uptake other soil bacteria (Dedysh et al., 2012). of ammonium seems likely as all genomes described to date appear to contain genes for the ammonia transporter channel (Amt) family (TC 1.A.11). Nitric oxide reductase genes (norB NITROGEN METABOLISM but not norC) were identified in ‘Koribacter versatilis,’ ‘Solibacter usitatus’ and Geothrix fermentans genomes. Genes encoding Nitrite reduction was observed in all three genomes reported dinitrogenase, a heterotetramer of the proteins NifD and NifK in 2009, and nitrate reduction in two of the initially analyzed (genes nifD and nifK, respectively) and dinitrogenase reductase,

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FIGURE 3 | Enzymes encoding genes of polysaccharide degradation (EC.3.2-) in different Acidobacteria subdivisions (GP) genomes. The comparisons were done using IMG: the integrated microbial genomes database and comparative analysis system.

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FIGURE 4 | Families of transporters (TC:3.A) in different Acidobacteria subdivisions (GP) genomes. The comparisons were done using IMG: the integrated microbial genomes database and comparative analysis system.

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a homodimer of the protein NifH (gene nifH), were found et al., 2012b) to 9.18 % in Granulicella mallensis type strain only in the genome of H. foetida. Ammonia monooxygenase MP5ACTX8T (Rawat et al., 2013). An overview of transporter (amo) and nitrous-oxide reductase (nosZ) genes were not found families in 10 acidobacterial genomes is provided in Figure 4. in any of the available genomes. Contrary to the previous The majority of transporters found in acidobacterial genomes report (Ward et al., 2009), homologs of nitrate reductases narB belong to the Drug/Metabolite transporter superfamily. The high and narG were not found in available genome sequences via number of different transport systems facilitates the acquisition in silico genome comparison, and physiological testing would of a broad range of substrate categories, including amino acids, therefore be required to suggest this function in Acidobacteria. peptides, siderophores, cation, or anions. The presence of a In summary, there is no clear evidence for the involvement of broad substrate range of transporters for nutrient uptake suggests Acidobacteria in key N-cycle processes such as nitrogen fixation, an advantage of Acidobacteria in complex environments and nitrification, or denitrification. adaptation to oligotrophic conditions, such as nutrient-limited soil conditions (Figure 4). Although iron metabolism and iron transporters were EXOPOLYSACCHARIDES discussed in genome sequence exploration studies, these characteristics have not been unequivocally demonstrated Exopolysaccharide (EPS) production has frequently been in culture-based studies. The only direct indication is the reported in cultured Acidobacteria species (Eichorst et al., 2007; observation of iron accumulation in B. elongata (Dedysh Pankratov and Dedysh, 2010; Whang et al., 2014). Initial genomic et al., 2012). Based on genome content, we speculate that analyses revealed that at least one Acidobacteria encodes genes the representatives of subdivisions 4, 8 and B. aggregatus involved in the EPS biosynthesis, specifically for the production may also be able to uptake ferric iron, and genes encoding of bacterial cellulose, genes involved in the cellulose synthesis transport systems involved in translocation across the outer are encoded on genomes belonging to subdivision 1 (exception and cytoplasmic membranes, i.e., cobalamin/Fe3+-siderophore ‘K. versatilis’ Ellin345). However, it is not known if this is a uptake transporters and Fe3+ hydroxamate transporters, have general characteristic of the phylum Acidobacteria. It has been been identified in these genomes. Genomic analyses also suggest suggested that EPS-producing bacteria may be able to survive that Acidobacteria may release siderophores to scavenge iron for long periods in soil due to the protection provided by their from soil minerals by formation of Fe3+ complexes that can be EPS. The dominance of Acidobacteria in acidic environments taken up by those transporters or use siderophores from other and its resistance to pollutants like uranium (Ellis et al., 2003; microorganisms. Gremion et al., 2003; Barns et al., 2007), petroleum compounds (Abed et al., 2002), linear alkylbenzene sulfonate (Sanchez- Peinado et al., 2010) and p-nitrophenol (Paul et al., 2006) may ECOLOGICAL INFERENCES DERIVED therefore be related to the ability to produce large amounts FROM METAGENOMIC APPROACHES of EPS. The functions of EPS in soil are numerous. It may be involved Large genome fragments recovered by metagenomics may in the formation of the soil matrix, may serve as a water and contain intact metabolic pathways for mining ecologically nutrition trap, and may be involved in bacterial adhesion that relevant genome fragments from the environment. The first can facilitate soil aggregate formation (Eichorst et al., 2007, acidobacterial metagenomic insert was described by Liles et al. 2011; Pankratov and Dedysh, 2010). Based upon the presence (2003). From a bacterial artificial chromosome (BAC) library, of cellulose synthesis genes and a large number of novel high- 12 (out of 24,400) clones were identified as containing molecular-weight proteins with excretion pathway motifs, it has acidobacterial 16S rRNA sequences (nine clones from subdivision been postulated that many Acidobacteria have the potential to 6, two from subdivision 4, and one from subdivision 5), and one form biofilms, resist desiccation and facilitate soil aggregate clone affiliated with subdivision 5 was selected for full sequencing. formation. However, to date, there are still no physiological Up to date there is no representative isolate available for this studies demonstrating actual acidobacterial EPS production or subdivision 5. The annotation of 20 ORFs revealed genes involved demonstration of its ecological role. in cell cycling, cell division, folic acid biosynthesis, DNA repair, and an ABC transporter. In addition, a novel 1,4-butanediol diacrylate esterase gene was found with 40% sequence identity to TRANSPORTERS an esterase from Brevibacterium linens. This enzyme is known to catalyze the conversion of insoluble butanediol diacrylate to a Acidobacteria have a large proportion of genes encoding for hydrolyzed soluble form for the use as a carbon source, suggesting transporters (Challacombe et al., 2011). The comparison of that the bacterium containing this fragment may possess this three acidobacterial genomes (two Acidobacterium capsulatum capability. subdivision 1 and one Ellin6076 subdivision 3) showed about 6% Another six acidobacterial genomic fragments (four out of six of the total coding sequences are transporters (Ward et al., 2009). related to subdivision 6) were recovered from a sandy ecosystem The Carbohydrate transport and metabolism category given by (Quaiser et al., 2003). Interestingly, two of the recovered clones cluster of orthologous groups (COG) classification can range affiliated with subdivision 6 contained regions of homology from 8.6% in Terriglobus saanensis type strain SP1PR4T (Rawat encoding a tyrosyl-tRNA synthetase, a metal-depending protease,

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FIGURE 5 | The Acidobacteria subdivisions phylogenetic tree. The phylogenetic tree is based on 220 sequences of 26 different subdivisions (GPs) of Acidobacteria from Silva database (http://www.arb-silva.de/) classified by RDP classifier. The sequences were aligned in Clustal X12 and the selection of conserved blocks from multiple alignment was carried out by Gblocks (Talavera and Castresana, 2007) for phylogenetic analysis. The phylogenetic tree was based on Neighbor-Joining clustering algorithm with 1000 bootstrap. Circles in the branches of the tree represent bootstrap support more than 75%. Outgroup is Castenholzii roseiflexus.

as well as eight or nine purine biosynthesis proteins. In a gene revealed that nine out of 20 ORFs were related to sequences subsequent examination of fosmid libraries from deep sea derived from members of Acidobacteria. Similarly, genes sediments (Quaiser et al., 2008), recovered this same syntenic involved in polyketide synthesis pathways were identified from region in eight out of 11 acidobacterial genome fragments a fosmid library in cloned inserts containing genes significantly affiliated with subdivision 6. In a metagenomic study of similar to genes from ‘S. usitatus’(Parsley et al., 2011). Primers Acidobacteria in a former agricultural soil, an additional four out targeting the mtaD homolog (encoding protein involved in of 17 fosmids (from a library of 28,800 clones) were recovered myxothiazol biosynthesis) identified in a metagenomic library with the same genomic region (Kielak et al., 2010). Thus, it were also used to screen acidobacterial isolates. In four out of six appears that a large percentage of the subdivision 6 Acidobacteria isolates examined (belonging to subdivisions 3, 4, and 6) mtaD members present in both terrestrial and marine environments, homologous sequences were identified, suggesting widespread contain this conserved genomic region adjacent to their rRNA distribution of PKS pathways among Acidobacteria (Parsley et al., operons. However, the ecological and evolutionary significance 2011). Genes involved in PKS biosynthesis were also identified of this striking pattern remains unknown. in sequenced acidobacterial genomes (Ward et al., 2009). The In a study designed to recover genes encoding the synthesis of only report of antibacterial metabolites in relation to genomic N-acyl homoserine lactones (NAHL), Riaz et al.(2008) identified potential was described by Craig et al.(2009), who isolated and a qlcA gene with a lactonase activity for the degradation of characterized metabolites derived from acidobacterial genome NAHLs. Sequencing of the genomic fragment containing this fragments hosted by Ralstonia metallidurans. These compounds

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showed inhibitory activity against E. coli, Bacillus subtilis, and nutrient cycling and plant growth after drastic disturbance Staphylococcus aureus. However, further analysis revealed that (Huang et al., 2015). the novel metabolites reported by these authors appeared to arise A number of studies have compared acidobacterial from a mixed biosynthetic pathway involving both the type III distribution and diversity in relation to proximity to plant polyketide synthase encoded by acidobacterial genome fragments roots or plant exudates. Numerous studies based on 16S rRNA and endogenous R. metallidurans enzymes. Multiple reports on sequences have shown a higher proportion and diversity of PKS pathways identified in Acidobacteria suggest widespread Acidobacteria in the bulk soil as compared to in the rhizosphere distribution of such pathways across this phylum, suggesting a (Marilley and Aragno, 1999; Sanguin et al., 2006; Fierer et al., role in the persistence, resistance and abundance of these bacteria 2007; Singh et al., 2007; Kielak et al., 2008). Other studies in soil ecosystems. However, without the characterization of have shown Acidobacteria abundant in red pepper (Capsicum the polyketide products, the production of antimicrobials by annuum L.) rhizosphere (Jung et al., 2015). Additionally, it was Acidobacteria remains speculative. shown by means of shotgun metagenomics that Acidobacteria is A moderately thermostable lipase (optimum temperature overrepresented in the soybean rhizosphere as compared to the between 50 and 60◦C) from a member of Acidobacteria phylum bulk soil (Mendes et al., 2014). was also described by metagenomic approach from forest In several cases, Acidobacteria have appeared to tolerate soil (Faoro et al., 2012). Interestingly, phylogenetic analysis various pollutant such as PCBs and petroleum compounds, linear revealed that the lipase-encoding gene was of fungal origin alkylbenzene sulfonate, p-nitrophenol, (Abed et al., 2002; Paul and was acquired via horizontal gene transfer. In total, large- et al., 2006; Sanchez-Peinado et al., 2010) and heavy metals insert metagenomics analyses have served to recover numerous (Ellis et al., 2003; Gremion et al., 2003; Barns et al., 2007) acidobacterial genomic regions, but the actual ecological insights leading to speculation that Acidobacteria may be involved in the offered from such fragmented datasets remain limited. degradation of certain pollutants. However, although the relative abundance of Acidobacteria has often been correlated to specific pollutants, no data, up to date have been reported that support ENVIRONMENTAL SURVEYS THAT actual activities related to pollutant degradation. Such pollutants appear to have little effect on acidobacterial diversity levels. CORRELATE Acidobacteria For example, uranium-contaminated soil possessed an extremely DISTRIBUTION WITH ENVIRONMENTAL broad diversity of acidobacterial populations, and these soils were FACTORS OR CONSTRAINTS actually the basis for the expansion of acidobacterial subdivisions to 26 (Barns et al., 2007). Given the high diversity within the Acidobacteria phylum, as With the broader characterization of subdivisions and well as within particular subdivisions (Figure 5), it is expected increasing depth of coverage it has now become possible to that they also represent a wide range of physiological traits, break down analyses to the subdivision level, which can be as observed for other highly abundant and diverse bacterial far more informative. The available studies provide distribution phyla, such as the Proteobacteria. However, most of the studies patterns of different acidobacterial subdivisions across different up to date focus on Acidobacteria at phylum level leading environmental gradients such as pH, nutrients, and carbon. to gross generalizations that may not mirror the ecological Especially striking is the predominance of Acidobacteria in low traits representative of lower taxonomic levels. Nevertheless, pH conditions, in particular members from subdivision 1 (Sait some general trends have been discerned from such broad- et al., 2006). On the other hand, Barns et al.(1999) suggested level analyses. In the most expansive study conducted to date, that some acidobacterial subdivisions had an aversion to low pH pyrosequencing of 16S rRNA gene fragment was used to examine conditions in soil and other case, subdivision 6 can be either the biotic or abiotic factors that most influence the abundance, positively or negatively correlated with soil pH (Chan et al., 2006; diversity and composition of soil acidobacterial communities Mukherjee et al., 2014). The abundance of subdivisions 1, 2, 3, 12, in different types of soils (88 types; Jones et al., 2009). Fierer 13, 15 was negatively correlated with soil pH, yet subdivisions 4, et al.(2007) studies have shown, based upon 16S rRNA gene 6, 7, 10, 11, 11, 16, 17, 18, 22, 25 showed a positive correlation. sequence distributions across 71 soils differing in geochemical Similarly, terminal restriction length polymorphism (T-RFLP) characteristics, that the abundance of Acidobacteria was generally and cultivation strategies detected a significant decrease of higher in soils with very low resource availability (low C Acidobacteria from subdivisions 1, 2, and 3 at soil pH higher than mineralization rate) and that the proportions of Acidobacteria 5.5 (Männistö et al., 2007). This trend appears also to hold for were higher in C-poor bulk soils in comparison to the cold-adapted populations of Acidobacteria (Lipson and Schmidt, rhizosphere. However, this position has been challenged by 2004; Männistö et al., 2007). Similar trends have been found in Jones et al.(2009) and Navarrete et al.(2013b) who found other environments as well, and Acidobacteria from acid mine a positive correlation between acidobacterial abundance and drainage systems seem to be even better adapted to more acidic organic carbon availability. At the phylum level, many studies conditions (pH 2–3) than Acidobacteria from soil environments have shown that Acidobacteria is sensitive to inorganic and (Kleinsteuber et al., 2008). Furthermore, it has been observed organic nutrients inputs (Cederlund et al., 2014; Koyama et al., that the phylogenetic diversity of acidobacterial communities 2014; Pan et al., 2014; Navarrete et al., 2015) and Acidobacteria becomes increasingly constrained as soil pH deviates from seemed to have a role in recovering soils as beneficial to soil neutrality (Jones and Martin, 2006). A possible explanation

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may lie in increased cell specialization and enzymes stability and 6 correlated with Mg and Ni contents present in serpentine at more extreme pH, where closely related Acidobacteria may tropical savanna soil. Additionally, da Rocha et al.(2013) showed share similar cellular strategies to deal with discrepancies between that the Holophagae (subdivision 8) respond to leek roots (qPCR intra- and extra-cellular pH. Despite this strong correlation with based study) either by increased cell division and/or by increase pH, it is not yet clear if this represents a direct causal relationship in genome quantity per cell. or if it is the result of other environmental factors that co-vary with pH. In fact, by measuring soil factors such as Al, Ca, Mg, K, ARE ACIDOBACTERIA OLIGOTROPHS? B, and micronutrients that had not been taken into account in previous studies, Navarrete et al.(2013b) demonstrated that The strong negative correlation between the abundance of subdivisions 4, 6, and 7 may actually respond to decreases Acidobacteria and concentration of organic carbon in soil has in soil aluminum and soil Ca and Mg in tropical soils. The led to the conclusion that members of this phylum may be subdivisions 6 and 7 responded to high contents of soil Ca, Mg, oligotrophic bacteria. However, it was pointed out that not Mn, and B, what elements are required for the growth of all living necessarily all members would be oligotrophic (Fierer et al., organisms. Magnesium ions are required by large number of 2007). In addition, genome sequences revealed the presence enzymes for their catalytic action, including all enzymes utilizing of only one or two copies of 16S rRNA genes suggesting or synthesizing ATP, or those that use other nucleotides to lower growth rates, which has been previously correlated with synthesize DNA and RNA. However, the ionic magnesium cannot oligotrophy (Klappenbach et al., 2000; Stevenson and Schmidt, directly be up taken by the biological membranes because they 2004; Eichorst et al., 2007; Kleinsteuber et al., 2008; Ward et al., are impermeable to magnesium (and other ions), so transport 2009). It is important to mention that although cultivation proteins must facilitate the flow of magnesium and other ions, strategies regarding Acidobacteria isolates are often referring to both into and out of cells (Beyenbach, 1990). Future studies nutrient limiting media (Janssen et al., 2002; Stevenson et al., on Acidobacteria subgroups 4, 6, and 7 functions certainly 2004; Davis et al., 2005) some of the isolates were shown to be will elucidate the role of those subgroups in soil. In addition, able to grow in higher carbon sources concentrations (Eichorst subdivision 10 has been shown to correlate with soil factors et al., 2007; de Castro et al., 2013). linked to soil acidity such as pH, Al, and Al saturation while The two observations mentioned above (negative correlation subdivision 13 was correlated with soil P, B, and Zn (Navarrete of Acidobacteria with organic carbon and lower growth rates) et al., 2013b). The subdivision 2 has been reported in Amazonian are also consistent with the ecological role of K-strategists. It has forest soil (Navarrete et al., 2013b) and in Mata Atlantica and been predicted that K-strategists would prosper in environments Cerrado soils (Catao et al., 2014) correlated with Al3+ levels and with low abundance of nutrients, which is not the same as to say CO2 concentration. Aluminum in tropical and subtropical soils that they are oligotrophs (Andrews and Harris, 1986). Compared is toxic to crops (Yachi and Loreau, 1999). This might suggest to r-strategists, K-strategists are predicted to have lower growth that subdivisions 2 and 10 may have metabolic tolerance to rates, but high efficiency in converting nutrients to biomass aluminum in soil. Subdivision 4 abundance tends to increase with as well as high tolerance to toxic compounds, among other increasing soil pH, suggesting different physiologies for members characteristics. Microbes that present the ecological K strategy of this subdivision (Foesel et al., 2013). Thus, it is important to are better competitors in oligotrophic environments (Andrews treat soil pH as is a master variable that is related to additional and Harris, 1986). Although the term oligotroph has been used in changes in other soil factors, such as Al concentration and macro- different ways, it usually describes an organism that is not able to and micro-nutrient availabilities (McBride, 1994), which may grow or thrive in environments with high nutrient concentrations represent the actual drivers for observed microbial community (Koch et al., 2008). However, it may be premature to assume dynamics. that all Acidobacteria have the same ecological strategy, since Navarrete et al.(2013b) and Pessoa-Filho et al.(2015) showed metagenomic data and physiological description of different that different subdivisions showed disparate correlations with subgroups have indicated that there is a high variation within this respect to soil nutrient or chemical status. Although subdivision phylum. 1 has negative correlations with P, C, and N, members of subdivisions 5, 6, and 17 appeared to be high abundant in more nutrient-rich soils. Similarly, Männistö et al.(2012) observed ACIDOBACTERIAL INTERACTION WITH phenotype dependent responses (within members of subdivisions OTHER MICROBES 1 and 2) to seasonal changes in Arctic tundra soil ecosystem, which were related to nutrient and carbon availability. Foesel Additional evidence for interaction with soil bacteria is the fact et al.(2013) provided culture-independent evidence for a distinct that Edaphobacter aggregans and B. elongata were isolated from niche specialization of different Acidobacteria, even from the co-cultures with methanotrophic bacteria. It was demonstrated same subdivision, due to particular soil physicochemical (pH, that B. elongata was unable to use CO2 and other C1 carbon temperature, nitrogen or phosphorus) and biological parameters compounds, which would be produced by the methanotrophic (respiration rate, abundances of ciliates or amoeba, vascular plant partner. Instead, it was proposed that the Acidobacteria was using diversity) in grassland and forest soils. Pessoa-Filho et al.(2015) the exopolysaccharides produced by the methanotroph as carbon showed the natural dominance of acidobacterial subdivisions 4 source (Koch et al., 2008; Dedysh et al., 2012).

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It is suggested that there is ecological relationship between The 16S rRNA data provided by next generation sequencing Acidobacteria and Proteobacteria because they are often observed together with soil chemicals (macro and micronutrients) can help to be intimately associated with each other in the environment, to elaborate specific culture medium for different Acidobacteria and may influence each other’s position in the community. subdivisions isolation. Despite the limitation of current genome- Meisinger et al.(2007) observed, via Fluorescence in situ based studies, the genomes obtained to date still give important hybridization (FISH) counts, that members of subdivisions 7 and hints related to the factors that explain the successful adaptation 8 were always associated with epsilon or gamma-proteobacteria of this phylum to harsh soil conditions. These factors include in filamentous microbial mats in hydrogen sulfide-containing the large number of high-affinity transporters, the potential springs. It was therefore hypothesized that the Acidobacteria utilization of a wide variety of carbohydrates as substrate, the often live as chemo-organotrophs in association with the resistance to antibiotics and production of secondary metabolites, autotrophically fixed carbon in the poorly oxygenated regions. the production of EPS and the potential use of bacterial produced Enrichment strategies have also often recovered consortia polymers such as gellan gum. comprised of Acidobacteria and Proteobacteria, as exemplified Although little direct evidence, genomic studies reveal that by the co-cultivation of subdivision 6 members from freshwater decomposition and utilization of natural polymers such as chitin, lake sediments with Alphaproteobacteria (Spring et al., 2000). cellulose, EPS, and gellan gum as potential important aspects However, it is not yet clear if co-cultivation stems from for future studies. Also better knowledge about the production overlapping niches between the different consortium members of EPS, biofilm, and secondary metabolites in Acidobacteria or if they have necessary metabolic interactions. It has been subdivisions is of importance to understand the survival, suggested that co-cultures containing acidobacteria should be resistance, persistence in soil as well as possible interactions of studied more closely to reveal potential ecological interactions members of this phylum with other soil microorganisms. As and growth preferences (Stevenson et al., 2004). Also, given Acidobacteria are ubiquitous, they should interact, positively or advances in sequencing, such enrichment cultures should be negatively, with other soil habitants. Therefore, unraveling these able to yield full genome sequences of a much broader range of interactions is vital for the proper understanding of their role in acidobacteria that available in pure culture. Certain groups of terrestrial ecosystem functioning. Proteobacteria have been associated with copiotrophic lifestyles Additionally, the recovery of 16S rRNA genes from the and given this association, Smit et al.(2001) hypothesized that environment should be taken forward. These surveys have the ratio between Proteobacteria and Acidobacteria (P/A) may provided new insight in terms of distribution of different provide insight into the general nutrient status of soils. Low P/A acidobacterial subdivisions and relation to environmental ratios would be indicative of oligotrophic soils, while high ratios variables, but more experimental approaches need to be coupled would be observed under copiotrophic conditions. with high throughput toolbox to tease out the actual roles of environmental variables. Further, more molecular studies that attempt to look at activities such as metatranscriptomic and stable CONCLUSION AND FUTURE isotope probing (SIP) approaches might be considered. DIRECTIONS

The high abundance and ubiquity of Acidobacteria in soils raises AUTHOR CONTRIBUTIONS questions related to the physiological traits that have led to this marked success. Although genome sequences have provided Analyzed the data: AK, CB, and EK. Contributed reagents/ important information, our integration of genomes has often not materials/analysis tools: JvV and EK. Wrote the paper: AK, CB, been informed by studies, and genomics analyses remain highly GK, JvV, and EK. skewed Acidobacteria subdivision 1, the groups for which most cultures are available. There is therefore an urgent need to isolate ACKNOWLEDGMENTS and sequence genomes of representatives from other subdivisions in order to understand their basic characteristics. Due to the still This work was financially supported by the Dutch Ministry of problematic cultivation of Acidobacteria, techniques like micro- Economic Affairs, Agriculture and Innovation (729.004.016) and cultivation and single-cell sequencing should give steps forward Brazilian Science Without Borders, Special Visiting Researcher to obtain a more representative range of acidobacterial genomes. (88881.062152/2014-01) financed by CAPES (Coordination In addition, with the increased high throughput of shotgun for the improvement of Higher Education Personal, Brazil). metagenomic studies and associated postgenomic, it should Publication 6078 of the Netherlands Institute of Ecology (NIOO- be possible to start dissecting acidobacterial genomes from KNAW). environmental datasets, thereby circumventing the necessity for cultivation. As an intermediate step, metagenomic analyses of more simplified systems, such as enrichment and non- SUPPLEMENTARY MATERIAL axenic cultures should also yield access to important genome information. It must, however, be stressed that cultivation efforts The Supplementary Material for this article can be found online remain a top priority, as these provide the necessary material for at: http://journal.frontiersin.org/article/10.3389/fmicb.2016. physiological studies and confirmations of genomic predictions. 00744

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Frontiers in Microbiology| www.frontiersin.org 16 May 2016| Volume 7| Article 744